Natural Killer (NK) cells are the first line of defense against infection. During their lifetime, NK cells undergo dynamic processes: as they develop, NK cells commit, proliferate, differentiate, and then arrest. Once activated, they immediately acquire cytotoxicity and become quite rapacious in killing target cells. NK cells are predisposed to killing because they are programmed for that function during their development. Our long-term goal is to understand the regulatory mechanisms controlling NK cell dynamics as a prerequisite to the development of successful NK-cell based adoptive immunotherapy. Although the collective efforts, accumulated for the last 20 years, resulted in the identification of key positive regulators of NK cells, the current knowledge failed to identify or elucidate the mechanisms of NK cell negative regulation. We searched for potential candidates and successfully identified TGF? as a potent negative regulator of NK cells1. To date, TGF? remains the only identified negative regulator of NK cell number. Therefore, it is imperative that the mechanisms by which TGF? operate in NK cells are understood. The specific hypothesis driving the proposed research is that TGF? limits the production and restricts the cytotoxicity acquisition of NK cells in response to viral infection. This hypothesis is based on three preliminary data: First, lack of TGF?R signaling results in augmented production of NK cells at terminal differentiation in the bone marrow. Second, lack of TGF?R signaling revealed a previously undescribed population of Granzyme Blow NK cells which is otherwise suppressed in normal mice. Third, mice having TGF?-resistant Granzyme Blow NK cells are resistant to murine cytomegalovirus. Using a combination of in vitro and in vivo approaches, we will test our hypothesis in two Specific Aims: In Aim 1, we will determine the mechanisms by which TGF? controls the production of NK cells. Two studies are designed to investigate i)-how TGF? regulates the processes of differentiation, survival, and proliferation during the production of NK from the bone marrow, and i)-why NK cells at their terminal differentiation are particularly susceptible to TGF? signaling. One prerequisite for the development of successful NK-cell-based immunotherapy in the future is to determine the factors that condition the survival, expansion, and self renewal of NK cells. The possibility that a lack of TGF?R signaling can prolong NK cell survival will support the notion that blocking the TGF?R pathway in NK cells prior to adoptive transfer into patients is a possibility for improving the design of NK-cell-based immunotherapeutic strategies. In Aim 2, we will determine the mechanism by which TGF? restricts functional competence of NK cells during response infection. Three studies were designed to determine direct outcomes of a lack of TGF?R signaling on i)- acquisition of cytotoxicity, ii)-clonal expansion and contraction in response to viral infection, and iii)-tolerance to viral ligands. We believe that understanding how negative regulation by TGF? restricts NK cell cytotoxicity will provide a strategy to make NK cells stronger in facing evolving viruses. PUBLIC HEALTH RELEVANCE: A type of white blood cells called Natural Killer (NK) cells may hold the key to combating infection. First discovered in the 1980's, NK cells are recognized as the immune system's front-line defense against infection. Circulating through the body by way of the blood and lymph systems, the majority of NK cells present in the body are in a resting state. Once activated, NK cells become quite rapacious in their search-and-destroy activities. Upon encountering a target cell (an infected cell or cancer cell), the activated NK cell attaches to the membrane of the target cell and injects cytoplasmic granules that quickly dissolve (lyse) the target cell. In less than five minutes, the infected or cancer cell is dead and the NK cell moves on to its next target. A single NK cell can destroy up to 27 target cells before it dies. The importance of NK cells in combating infection is undisputable but viruses are cleaver because they have evolved mechanisms for modulating NK cell activity to persist in spite of an active host immune system. That means we need to develop stronger NK cells to face the evolving viruses. In this research, we propose to study the function of "super"-NK cells developed to be stronger in killing infected cells. These super-NK cells were generated by genetic blockage of TGF? signaling pathway which renders them resistant to TGF? action. They will be called "TGF?-resistant NK cells". Studying the "super" killing functions of TGF?-resistant NK cells is the major goal of this project. Overall, this comprehensive study has a strong potential of clinical applicability. It is novel because it is one of a very few studies to directly assess in vivo the role of TGF? in NK cells, and it is innovative because it uses a unique in vivo model. Outcomes of the project are expected to generate new insight into the fundamental knowledge of NK cells and likely will provide a promising strategy to improve current NK cell- based immunotherapies.